Host cells expressing recombinant human erythropoietin

ABSTRACT

The gene coding for human erythropoietin (EPO) was obtained from human genomic DNA. Thc gene used does not include sequences from regions at i 5′ of the first translated ATG and ii 3′ of the stop codon of the EPO gene. The gene was cloned into an expression plasmid for eukaryotic cells that have as sole expression control elements the early promoter of the SV40 virus and its polyadenylation signal. Recombinant cells resulting from transfection with genetic constructs used provide an unexpectedly high level of protein expression of 50 mg of recombinant EPO per liter of culture medium per day.

This Application claims benefit under 35 U.S.C . § 371 of InternationalApplication No. PCT/US99/26238, filed on Nov. 8, 1999, which waspublished under PCT Article 21(2) in English and which is fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates, in general, to a host cell and a vectorcomprising a nucleotide sequence coding for recombinant humanetythropoietin (EPO). In particular, the expression vector comprisesonly one promoter that regulates the EPO expression. The presentinvention also refers to a method of producing EPO and the EPO thusproduced.

2. Background Information

EPO is a glycoprotein that stimulates erythroblast differentiation inthe bone marrow, thus increasing the circulating blood erythrocytecount. The mean life of erythrocytes in humans is 120 days andtherefore, a human being loses 1/120 erythrocytes each day. This lossmust be continuously restored to maintain an adequate level of red bloodcells.

The existence of EPO was first postulated by the turn of the century andwas definitely proved by Reissman and Erslev early in the '50s. SeeCarnot, et al., C.R. Acad. Sci. (France)143:384-386 (1906); Carnot, etal., C.R. Acad. Sci. (France), 143:432-435 (1906); Carnot, et al., C.R.Soc. Biol., 111:344-346 (1906); Carnot, C.R. Soc. Biol., 111:463-465(1906); Reissman, Blood, 5:372-380 (1950) and Erslev, Blood 8:349-357(1953). Reissman and Erslev's experiments were promptly confirmed byother researchers. See Hodgson, et al., Blood, 9:299-309 (1954); Gordon,et al., Proc. Soc. Exp. Biol. Med., 86:255-258 (1954) and Borsook, etal., Blood, 9:734-742 (1954).

The identification of the EPO production site in the organism was anissue of debate. Successive experiments led to the identification of thekidney as the main organ and peritubular interstitial cells as thesynthesis site. See Jacobson, et al., Nature, 179:633-634 (1957);Kuratowska, et al., Blood, 18:527-534 (1961); Fisher, Acta Hematol.,26:224-32 (1961); Fisher, et al., Nature, 205:611-612 (1965); Frenkel,et al., Ann. N.Y. Acad. Sci., 149:292-293 (1968); Busuttil, et al.,Proc. Soc. Exp. Biol. Med, 137:327-330 (1971); Busuttil, Acta Haematol.,(Switzerland), 47:238-242 (1972); Erslev, Blood, 44:77-85 (1974); Kazal,Ann. Clin. Lab. Sci., 5:98-109 (1975); Sherwood, et al., Endocrinology,99:504-510 (1976); Fisher, Ann. Rev. Pharmacol. Toxicol., 28:101-122(1988); Jelkmann, et al., Exp. Hematol., 11:581-588 (1983); Kurtz, etal., Proc. Natl. Acad. Sci. (USA), 80:4008-4011 (1983); Caro, et al., J.Lab. Clin. Med., 103:922-931 (1984); Caro, et al., Exp. Hematol., 12:357(1984); Schuster, et al., Blood, 70:316-318 (1986); Bondurant, et al.,Mol. Cell. Biol., 6:2731-2733 (1986); Schuster, et al., Blood,71:524-527 (1988); Koury, et al., Blood, 71:524-527 (1988); Lacombe, etal., J. Clin. Invest., 81:620-623 (1988); Koury, et al., Blood,74:645-651 (1989).

A smaller proportion, ranging from 10% to 15% of total EPO, is producedby the liver in adults. See Naughton, et al., J. Surg. Oncol.,12:227-242 (1979); Liu, et al., J. Surg. Oncol., 15:121-132 (1980);Domfest, et al., Ann. Clin. Lab. Sci., 11:37-46 (1981); Dinkelaar, etal., Exp. Hematol., 9:796-803 (1981); Caro, et al., Am. J. Physiol.,244:5 (1983); Dornfest, et al., J. Lab. Clin. Med., 102:274-285 (1983);Naughton, et al., Ann. Clin. Lab. Sci., 13:432-438 (1983); Jacobs, etal., Nature, 313:806-810 (1985); Erslev, et al., Med. Oncol. Tumor.Pharmacother., 3:159-164 (1986). The EPO produced is directlyproportional to the extent of tisular hypoxia and its expression risesby increasing the number of the EPO producing cells.

EPO has shown great efficiency in the treatment of anemia, especiallyanemia derived from renal failure. See Eschbach, et al., N. Enigland J.of Med., 316:73-78 (1987); Krane, Henry Ford Hosp. Med. J., 31:177-181(1983). Its therapeutical usefulness, however, has been limited due tothe unavailability of a massive production method. The quantity andquality of the EPO obtained by the extractive systems known wereinsufficient. Recently, the use of recombinant DNA technology has madeit possible to obtain large amounts of proteins. The application ofthese techniques to eukaryotic cells has allowed a large-scaleproduction of EPO. See patents U.S. Pat. No. 5,688,679 (to Powell), U.S.Pat. No. 5,547,933 (to Lin), U.S. Pat. No. 5,756,349 (to Lin), U.S. Pat.No. 4,703,008 (to Lin) and U.S. Pat. No. 4,677,195 (to Hewick et al.).

At the present, recombinant DNA techniques are widely known and used.These techniques involve the use of genetic elements such as DNAfragments and enzymes to assemble and transfer genetic constructions forthe production of recombinant proteins. The recombinant DNA techniquesalso facilitate the study of biological mechanisms. SeeFrank-Kamenetskii, “Unraveling DNA” [Samaia Glavnaia Molekula] (AddisonWesley Longman Inc., Reading, Mass., 1997); Brown, “Gene Cloning”(Chapman & Hall, London, England, 1995); Watson, et al., “RecombinantDNA”, 2nd Ed. (Scientific American Books, New York, N.Y., 1992); Albertset al., “Molecular Biology of the Cell” (Garland Publishing Inc., NewYork, N.Y., 1990); Innis et al., Eds., “PCR Protocols. A Guide toMethods and Applications” (Academic Press Inc., San Diego, Calif.,1990); Ehrlich, Ed., “PCR Technology. Principles and Applications forDNA Amplification” (Stockton Press, New York, N.Y., 1989); Sambrook etal., “Molecular Cloning. A Laboratory Manual” (Cold Spring HarborLaboratory Press, 1989); Bishop et al., “Nucleic Acid and ProteinSequence. A Practical Approach” (IRL Press 1987); Reznikoff, Ed.,“Maximizing Gene Expression” (Butterworths Publishers, Stoneham, Mass.,1987); Davis et al., “Basic Methods in Molecular Biology” (ElsevierScience Publishing Co., New York, N.Y., 1986); Watson, “The DoubleHelix” (Penguin Books USA Inc., New York, N.Y., 1969).

SUMMARY OF THE INVENTION

The claimed invention comprises an eukaryotic cell line that producesrecombinant human EPO, obtained by means of its transfection with anexpression vector that comprises a gene coding for human EPO. The vectorfurther comprises an unique promoter and terminator as expressioncontrol elements. SEQ ID NO:1 identifies the EPO amino acid sequencecodified by the gene used.

The invention provides a host cell comprising a vector which comprises anucleotide sequence encoding the erythropoietin polypeptide consistingof the amino acid sequence in SEQ ID NO:1, a viral promoter and a viralterminator.

The invention further provides a method for producing an EPOpolypeptide, comprising culturing the above host cell under suchconditions that said polypeptide is expressed and recovered.

One of the advantages of this invention is that the EPO coding geneutilized does not include non-coding fragments of the 5′ and 3′ regions.However, the system claimed produces an unexpectedly high amount of EPO.

An additional advantage of this invention is the use of expressionvectors comprising only one promoter, which exhibit a high EPOproductivity. By utilizing the claimed method, it is possible to obtainmore than 50 mg of EPO per liter of cell culture per day, that is, overfive times the EPO production level claimed by the best method reportedso far utilizing one promoter.

The combination of the EPO coding gene claimed in this invention and asimple promoter showed, surprisingly, to operate efficiently, resultingin a stable EPO producing cell. The transfected cells yielded an amountof EPO comparable to, or even higher than, those reported using intheory more adequate, though more complex and difficult to manipulate,genetic constructions.

An additional advantage of the claimed invention is the cotransfectionwith two vectors that confer different resistance, thus simplifying andfacilitating the selection, genetic amplification and maintenance of thecotransfected EPO producing cells.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates polyacrylamide gel (SDS-PAGE) analysis of an EPOsample obtained following the method described after purification. Inlanes 1, 4 and 7, molecular weight markers were loaded. In lanes 2, 3, 5and 6, different amounts of pure EPO obtained according to the claimedprocedure were run. The purity of the product obtained and the apparentmolecular weight exceeding 30 kDa is coincident with the one reportedfor urinary human EPO as could be clearly observed.

FIG. 2 illustrates a Western blot analysis of an EPO sample obtainedaccording to the method described. Identity of the EPO produced isassessed, since it is recognized by a monoclonal antibody against humanEPO. In lanes 1 and 2, a human EPO standard and molecular weight markerswere loaded, respectively. EPO samples obtained according to the claimedmethod were loaded in lanes 3 to 5.

FIG. 3 shows a SDS-PAGE analysis of a pure EPO sample obtained accordingto the method described, and further treated with glycanases. Molecularweight markers were loaded in lanes 1, 4 and 8. Lanes 2 and 7 correspondto untreated EPO. In lane 3, O-glycanase treated EPO was loaded; thepresence of an O-glycosilation site is verified. In lane 5, N-glycanasepartially digested EPO was loaded. The presence of 3 N-glycosilatedmolecules with molecular weights as expected for EPO can be verified.Lane 6 was loaded with EPO digested with N-glycanase, and the expectedmolecular weight for the wholly deglycosilated protein was obtained.

FIG. 4 illustrates a survey of the isoelectric points in pure EPOsamples produced according to the method described. EPO samples were runin lanes 2, 3 and 4, isoelectric point markers in lanes 1 and 5. Thepresence of isoforms corresponding to EPO are verified, showing anisoelectric point range of 3.0 to 4.5.

DETAILED DESCRIPTION OF THE INVENTION

The biological basis of recombinant DNA technology could be summarizedas follows.

DNA (deoxyribonucleic acid) is the genetic material of all living cellsand some viruses. Polymeric chains of four different nucleotides formthe DNA, each of them being a purine or pyrimidine bound to adesoxyribose. The sugar moiety is in turn linked to a phosphate group.These four nucleotides are: adenine (A), cytosine (C), guanine (G) andthymine (T).

The DNA chains are formed by phosphotriester linkages betweennucleotides, where the phosphate in position 5′ of the deoxyribose ofone nucleotide is bound to the 3′ position of the deoxyribose of theprevious nucleotide. Synthesis in vivo occurs from 5′ to 3′, which isthe conventional direction adopted to describe DNA sequences.

Functional DNA is presented as a double helix of complementary bases,where chains are held together by hydrogen bonds fomied between A's andC's of one chain and T's and G's of the complementary chain,respectively. This is the reason why they are referred to as “basepairs”.

The chains are also antiparallel, that is, the 5′ end of each helix ismatched to the 3′ end of the other, as depicted below:

5′-TACGTAC-3′

3′-ATGCATC-5′

For protein synthesis to occur certain DNA coding regions are firsttranscribed to messenger RNA (mRNA). The mRNA is translated in turn intoa protein. Each of the DNA coding regions is called a gene.

The synthesis of RNA (ribonucleic acid) chains involves thetranscription of certain gene regions by enzymes called RNA polymerases.An antiparallel RNA chain, complementary to the DNA template, is thusobtained. Each A from DNA will correspond to a U in the RNA, each C to aG, each G to a C and each T to an A. The RNA molecule is alsocharacterized because it is less stable than DNA. In addition, the sugarmoiety in RNA is ribose instead of desoxiribose as in DNA. RNA isfurther distinguished from DNA by the substitution of uracyl (U) inplace of thymine (T).

Matrix DNA 5′------------ ACGTAG ------3′ Synthesized mRNA3′------------ UGCAUC ------5′

In eukaryotic cells, synthesized mRNA is processed in the nuclei(splicing) to result in mature mRNA. This process is not verified inbacteria.

Mature mRNA is then taken as matrix to be translated into a protein, ina process where transfer RNA (tRNA, small RNA chains that carry aminoacids and align them specifically to form a protein) and ribosomes arethe main participants. Three mRNA bases (triplet or codon) code eachamino acid. For instance, the AUG sequence in mRNA codes for the aminoacid methionine. The mRNA chains are thus translated into a specificpeptide sequence, which finally folds into an active protein. Theprotein synthesis is called “expression.”

The amount of protein expressed depends, among other factors, on thepresence of certain DNA regions called promoters, which affect the rateat which the expression process occurs. In addition, there are DNAsequences that indicate the termination of transcription (terminators)and codons which indicate the end of translation (stop codons).

DNA technology involves the isolation of DNA fragments, either naturalor synthetic, and their insertion into cells (i.e. bacteria, yeast,insect and mammalian cells) to render them capable of producingheterologous proteins such as EPO. The proteins obtained by recombinantDNA technology are called recombinant proteins.

The application field of recombinant DNA technology is not limited tocultured cells, since genes can also be incorporated into multicellularorganisms (i.e. plants, insects, mammals and fish).

The expression of heterologous proteins requires the following elements:

A gene coding for the desired protein. The gene may be obtained usingdifferent techniques, such as;

Isolation from genomic DNA libraries.

In vitro synthesis of DNA chains. There are commercially availableequipments that synthesize relatively short DNA strands, making itpossible to synthesize a gene in vitro.

Amplification. Technology that allows to replicate several times a DNAfragment, such as a gene.

Others, i.e. as obtained from cDNA libraries synthesized from mRNA.

Active promoters to express a protein in the cell of interest.

Proper terminators so that transcription is correctly terminated.

Vectors. Genetic constructions such as plasmids or viruses that directthe gene with its promoter and terminator towards the inner region ofthe cell of interest incorporating the gene either in a chromosome orextrachromosomally. In certain cases, the incorporated gene may remainindefinitely in the cell and be transmitted to its progeny, or be lostin a relatively short term. There are multiple vector systems such asplasmids, and natural or modified viruses. It is also possible to usephysical means of DNA introduction such as cell or nucleimicroinjection. Viruses and plasmids are obtained from nature and aregenetically modified in vitro to achieve the desired characteristics.

Others. Additionally, other genetic elements may be necessary to improvethe selection of cells receiving the gene (i.e. another gene conferringresistance to antibiotics) or to amplify the number of copies of thegene in each cell (genetic amplification).

Vectors and genes should be as simple as possible to reduce the timenecessary to develop the system. A fundamental consideration is thatgenetic simplicity should not disregard the productivity or quality ofthe protein produced.

To achieve the expression of the protein of interest, the appropriatecorresponding gene is transfected with the proper vectors within thehost cell. Transfection may be done by different techniques such aselectroporation, precipitation with calcium phosphate and the use oflyposomes, among other techniques available.

The gene of interest may be associated to other genes already known toconfer resistance, for instance, to antibiotics such as geneticin, or totoxic agents such as methotrexate (MTX). This association allows theselection of the transfected cells in a stable manner, that is, thoseselected are capable of reproducing and transmitting the gene ofinterest to their progeny. Association also permits to select therecombinant cells showing the highest expression level of the protein ofinterest.

The recombinant product thus obtained is identified by its molecularweight, amino acid sequence and biological activity, among otherapplicable assays.

The tools (i.e. restriction enzymes) and techniques that gave rise torecombinant DNA technology were first developed in the early '70s andwere followed by an intense and widespread utilization. Moreparticularly, the genetic engineering techniques utilized presently toproduce EPO involve the following:

1. The use of EPO genes including fragments of non-coding regionslocated 5′ of the first translated ATG and 3′ of the stop codon of thegene. It is conventionally believed that the presence of expressioncontrol elements located in the non-coding regions of the gene isnecessary to achieve a high production of EPO. See patent U.S. Pat. No.5,688,679 (to Powell).

2. The employment of expression vectors with different promoters, wasbased upon the premise that a combination of promoters induces a higherEPO production. Until now, the use of only one promoter included in thevector has resulted in a low level of protein expression. See patentsU.S. Pat. No. 4,703,008 (to Lin), U.S. Pat. No. 4,677,195 (to Hewick etal.) and U.S. Pat. No. 5,688,679 (to Powell). Average production of EPOusing only one promoter is 200 μ/l/day. Maximum production of EPOreported using only one promoter is 10 mg/l/day.

3. The potential instability of the genetic systems due to thecomplexity of the genetic constructs utilized.

In order to obtain the claimed EPO producing cells, genomic DNA is firstextracted from human white blood cells. The EPO coding gene is obtainedfrom the isolated genomic DNA. To achieve this, the gene is amplifiedusing adequate primers to prevent the occurrence of 5′ and 3′ non-codingregions of the EPO gene. These primers include restriction sites intheir 5′ ends that remain at both ends of the isolated gene tofacilitate further cloning.

The amplified gene is next cloned in a bacterial vector and sequenced.Once the sequence obtained is verified, the gene is cloned into the XhoI-Hind III sites of an expression vector for eukaryotic cells harboringonly the SV40 early promoter and its terminator. The vector confersresistance to geneticin and ampicillin.

The CHO cells are subsequently cotransfected with two vectors: 1) theEPO expression vector and 2) a vector that confers resistance tomethotrexate. Stably transfected EPO producing cells are selectedaccording to their resistance to geneticin. The level of EPO expressionis monitored by the selection of amplified cells resistant to increasingconcentrations of methotrexate.

Finally, clones are selected according to their productivity level asmeasured by radioimmunoassay. Culture supernatants of the mostproductive clones are used to test the identity of the EPO produced bySDS-PAGE, Western blot, glycanase treatment followed by SDS-PAGE,isoelectric focusing and a complete protein sequence analyses. Thebiological in vivo activity of the produced EPO is determined by anex-hypoxic polycythemic mice assay using as reference the internationalstandard for EPO standard.

Vectors and Host Cells

The present invention relates to vectors which include a nucleotidesequence encoding EPO, host cells genetically engineered with therecombinant vectors, and the production of EPO polypeptides or portionsthereof by recombinant techniques.

Recombinant constructs may be introduced into host cells usingwell-known techniques such as infection, transduction, transfection,transvection, conjugation, electroporation and transformation. Thevector may be, for example, a phage, plasmid, viral or retroviralvector.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda P_(L) promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating codon (AUG or GUG) at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. More preferably, two expression vectors willinclude a total of two markers. Such markers include methotrexate,dihydrofolate reductase or neomycin resistance. Preferred vectors conferresistance to methotrexate and neomycin-derived antiobiotics such asgenetycin.

Especially preferred host cells are mammalian cells comprising CHO, COS,BHK, Namalwa and HeLa. Preferred host cells are CHO cells. Appropriateculture media and conditions for the above-described host cells areknown in the art.

A preferred method of obtaining EPO from the host cells of the inventionis culturing in media comprising insulin. Specifically, such culturingcomprises separating the supernatant which comprises EPO and insulinfrom the host cells of the invention, concentrating the supernatant andfreezing the concentrated product. Preferably, the culture mediacomprises between 0.5 mg and 20 mg insulin per liter of culture media.

Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Especially preferred vectors are pDHFR, and pVex 1. The pDHFR vectorincludes DNA encoding for the dehydrofolate reductase (DHFR) of mouse,whose expression is controlled by the early promoter of the SV40 virusand its polyadenylation signal. The pVex 1-EPO vector comprises the DNAencoding the EPO polypeptide in SEQ ID NO:1, an element conferringresistance to neomycin-derived antibiotics, an early promoter of theSV40 virus and the polyadenylation signal of the SV40 virus.

Suitable eukaryotic promoters for use in the present invention includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter. An especially preferredpromoter is the viral SV40 early promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., “Basic Methods in MolecularBiology,” (1986).

An especially preferred method to effect introduction of the constructof the invention into a host cell is by calcium phosphate transfection.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually of a size ranging from 10 to 300 bp that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at bp 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, either during purification, or duringsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification.

The EPO protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography.

A preferred method of purifying the EPO produced by the cells of theinvention comprises treating cell culture supernatants comprising EPO bya combination of the following steps: (a) differential precipitation,(b) hydrophobic interaction chromatography, (c) diafiltration, (d)anionic exchange chromatography, (e) cationic exchange chromatographyand (f) molecular exclusion chromatography. Preferably, said steps areperformed in the following order: (a), (b), (c), (d), (e), and (f).

A preferred method of using the EPO produced by the cells of the presentinvention comprises lyophilization into a form suitable for injectioninto humans for treatment of disease. Specifically, the preferredlyophilization procedure comprises placing the EPO into a pharmaceuticalcomposition, loading the first EPO composition into a container, whereinsaid container is at a temperature equal to or less than −30° C.;incubating said EPO composition at a temperature equal to or less than−30° C. under atmospheric pressure for a time equal to or greater than 4hours; incubating said composition at a pressure of equal to or lessthan 30 absolute microns for a time equal to or greater than one hour;and raising the temperature equal to or less than 3° C. per hour untilreaching at least 25° C., while keeping pressure values equal to or lessthan 5 absolute microns.

A preferred pharmaceutical composition for lyophilization comprises EPO,sugar, salts and human albumin. An especially preferred composition forlyophilization comprises EPO, mannitol, NaCl, NaH₂PO₄ and Na₂HPO₄.12H₂O.

Nucleic Acid Molecules

The host cells of the present invention may comprise vectors whichcomprise the EPO nucleic acid molecule from Lin, “DNA Sequences EncodingErythropoietins,” U.S. Pat. No. 4,703,008, which is herein incorporatedby reference, and variants thereof. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, ed. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the EPO protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are nucleic acid molecules encoding the EPO proteinhaving the amino acid sequence shown in SEQ ID NO:1.

Further embodiments of the invention include isolated nucleic acidmolecules comprising apolynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the EPO polypeptidehaving the complete amino acid sequence in SEQ ID NO:1 or (b) anucleotide sequence complementary the nucleotide sequence in (a).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 97%, 98%, or99% identical to a nucleic acid sequence encoding the EPO polypeptidewill encode a polypeptide “having EPO protein activity.” In fact, sinceall of each and every degenerate variant of these nucleotide sequencesencode the same polypeptide, this will be clear to the skilled artisan.It will be further recognized in the art that, for such nucleic acidmolecules that are not degenerate variants, a reasonable number willalso encode a polypeptide having EPO activity. This is because theskilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U., et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that preserve functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U., et al., supra, and the references cited therein.

The invention claimed is better explained by the examples depictedbelow:

EXAMPLES Example 1

Preparation of Human Genomic DNA

10 ml of blood were extracted from a clinically healthy human adult malesubject and added to a test tube containing 10 mM EDTA (pH 8). The bloodwas transferred in 5 ml aliquots to two 50 ml test tubes, to which 45 mlof a solution containing 0.3 M of saccharose, 10 mM Tris-HCl (pH 7.5), 5mM MgCl₂ and 1% Triton X 100 was added.

The solutions were then incubated on ice for 10 minutes and centrifugedfor 10 minutes at 1,000 g at 4° C. The supernatants were discarded andthe pellets rinsed several times with a 0.075 M NaCl solution containing0.025 M EDTA (pH 8), followed by centrifugation for 10 minutes at 1,000g at 4° C.

The resulting pellets thus obtained were resuspended in 3 ml of a 10 mMTris-HCl (pH 8), 400 mM NaCl, 2 mM EDTA (pH 8) solution. 200 μl of 10%SDS (sodium dodecyl sulfate) and 500 μl K proteinase (1 mg/ml in 1% SDSand 2 mM EDTA pH 8) were then added, and the solutions were incubatedovernight at 37° C. After the addition of 1 ml of NaCl saturatedsolution to each test tube the solutions containing the genomic DNA werecentrifuged at 2,500 g for 15 minutes.

Each supernatant was transferred to a 15 ml test tube where one volumeof isopropanol was added. The test tubes were gently mixed by inversionand stored at room temperature until a DNA precipitate was formed. Thegenomic DNA was then recovered with a hook-end Pasteur glass pipette.

The DNA was placed in a 2 ml test tube, and 1 ml of 70% ethanol wasadded. After one minute, the supernatant was discarded and theprecipitate was let dry. After drying, the precipitate was dissolved in500 μl of TE buffer (10 mM Tris-HCl pH 8-1 mM EDTA).

The concentration of the DNA solution was calculated by measuring theabsorbance of a 1:1000 dilution of the solution at 260 nm. It wasassumed that 50 μg of genomic DNA was equivalent to 1 OD unit. Asolution containing 500 ng of genomic DNA per μl of TE buffer wasprepared.

Example 2

Preparation of the EPO Construct

The EPO construct was prepared from 500 ng of human genomic DNA obtainedin Example 1. The following was added to 1 μl of the solution resultingfrom Example 1 placed on a 0.5 ml test tube: 400 ng of each of the EPO 1and EPO 2 primers, an aqueous solution of 2.5 mM of each deoxynucleotide(dATP, dCTP, dGTP and dTTP) and 2.5 units of Taq DNA polymerase (PerkinElmer) in a final volume of 100 l using the buffer recommended by themanufacturer. A thermal cycler was used and programmed for 30 cycles of:1 minute at 93° C., 1 minute at 55° C. and 3 minutes at 72° C. From thisreaction, a DNA fragment of approximately 2,170 base pairs containingthe EPO gene was obtained.

The nucleotide sequences of the primers utilized were as follows:

EPO 1: 5′ GAATTCTCGAGATGGGGGTGCACGGTGAG 3′ (SEQ ID NO:2). This primercorresponds to the first bases which were translated from the EPO genewith a site for the recognition of the Xho I enzyme and another site forthe recognition of the Eco RI enzyme in the 5′ end. These sites wereused in the subsequent cloning steps.

EPO 2: 5′ AAGCTTTCATCTGTCCCCTGTCCTGCA 3′ (SEQ ID NO:3). This primer iscomplementary to the last translated bases and the stop codon of the EPOgene. A site for the recognition of the Hind III enzyme was added to the3′ end of the primer. This site was used in subsequent cloning steps.

The nucleotide sequence obtained was as follows (SEQ ID NO:4):

gaattctcgagatgggggtgcacggtgagtactcgcgggctgggcgctcccgccgcccgggtccctgtttgagcggggatttagcgccccggctattggccaggaggtggctgggttcaaggaccggcgacttgtcaaggaccccggaaggggagggggtggggcagcctccacgtgccagcggggacttgggggagtccttggggatggcaaaaacctgacctgtgaaggggacacagtttgggggttgaggggaagaaggtttgggggttctgctgtgccagtggagaggaagctgataagctgataacctgggcgctggagccaccacttatctgccagaggggaagcctctgtcacaccaggattgaagtttggccggagaagtggatgctggtagctgggggtggggtgtgcacacggcagcaggattgaatgaaggccagggaggcagcacctgagtgcttgcatggttggggacaggaaggacgagctggggcagagacgtggggatgaaggaagctgtccttccacagccacccttctccctccccgcctgactctcagcctggctatctgttctagaatgtcctgcctggctgtggcttctcctgtccctgctgtcgctccctctgggcctcccagtcctgggcgccccaccacgcctcatctgtgacagccgagtcctggagaggtacctcttggaggccaaggaggccgagaatatcacggtgagaccccttccccagcacattccacagaactcacgctcagggcttcagggaactcctcccagatccaggaacctggcacttggtttggggtggagttgggaagctagacactgcccccctacataagaataagtctggtggccccaaaccatacctggaaactaggcaaggagcaaagccagcagatcctacggcctgtgggccagggccagagccttcagggacccttgactccccgggctgtgtgcatttcagacgggctgtgctgaacactgcagcttgaatgagaatatcactgtcccagacaccaaagttaatttctatgcctggaagaggatggaggtgagttccttttttttttttttccttttggagaatctcatttgcgagcctgattttggatgaaagggagaatgatcgggggaaaggtaaaaggagcagcagagatgaggctgcctgggcgcagaggctcacgtctataatcccaggctgagatggccgagatgggagaattgcttgagccctggagtttcagaccaacctaggcagcatagtgagatcccccatctctacaaacatttaaaaaaattagtcaggtgaagtggtgcatggtggtagtcccagatatttggaaggctgaggcgggaggatcgcttgagcccaggaatttgaggctgcagtgagctgtgatcacaccactgcactccagcctcagtgacagagtgaggccctgtctcaaaaaagaaaagaaaaaagaaaaataatgagggctgtatggaatacattcattattcattcactcactcactcactcattcattcattcattcattcaacaagtcttattgcataccttctgtttgctcagcttggtgcttggggctgctgaggggcaggagggagagggtgacatgggtcagctgactcccagagtccactccctgtaggtcgggcagcaggccgtagaagtctggcagggcctggccctgctgtcggaagctgtcctgcggggccaggccctgttggtcaactcttcccagccgtgggagcccctgcagctgcatgtggataaagccgtcagtggccttcgcagcctcaccactctcttcgggctctgggagcccaggtgagtaggagcggacacttctgcttgccctttctgtaagaaggggagaagggtcttgctaaggagtacaggaactgtccgtattccttccctttctgtggcactgcagcgacctcctgttttctccttggcagaaggaagccatctcccctccagatgcggcctcagctgctccactccgaacaatcactgctgacactttccgcaaactcttccgagtctactccaatttcctccggggaaagctgaagctgtacacaggggaggcctgcaggacaggggacagatgaaagctt

The first translated atg codon, as well as the tga “stop” codon, areunderlined. The sequences of restriction sites utilized in the cloningare shown in bold italics. It should be noted that more than one codonmay code for the same amino acid, and that consequently, the EPO proteincould be translated from different mRNA templates having differentnucleotide sequences but coding nevertheless for EPO.

Example 3

Cloning and Sequencing of the EPO Gene

A fragment of approximately 2,170 base pairs containing DNA coding EPOwas purified. The ends of the DNA were blunted by treatment with the DNApolymerase Klenow's fragment and cloned in the Sma I site of a M13mp18vector, following standard molecular biology techniques. The recombinantplasmids obtained were cut with Xho I and Hind III enzymes. The presenceof the insert was verified by electrophoresis of the restrictionfragments in a 0.8% agarose gel stained with ethydium bromide. Apositive clone (two bands, one having approximately 2,200 base pairs andthe other one corresponding to the linearized vector) were selected. Theinserted EPO gene was sequenced according to Sanger's technique using aT7 sequencing kit (Pharmacia). Some regions of the EPO gene were furthersequenced with an automatic 370 A Applied Biosystems Internationalsequencer. For each sequencing system the protocols recommended by themanufacturers were followed.

Example 4

Vectors for Eukaryotic Cells

1. Construction of pVex 1 Vector

The pVex1 vector was built following the standard molecular biologyteclniques. It consisted of:

a. Fragments of the bacterial pBR322 vector, which have a bacterialreplication origin and confer resistance to ampicillin, foramplification and selection of the vector in E. coli.

b. Immediately downstream from a) follows an early promoter of the SV40virus, which allowed the expression of the genes cloned at 3′ from thiselement.

c. Immediately downstream from b) follow the Xho I and Hind III cloningsites, which allowed the insertion of the genes to be expressed.

d. Immediately downstream from c) follows the polyadenylation signal ofthe SV40 virus, which allowed the proper polyadenylation of the specifictranscripts of the gene cloned in c).

e. Immediately downstream from d) follow the TK promoter and the genecoding for neomycin phosphotransferase with its polyadenylation signal.These elements allowed the selection of stably transfected cells throughthe use of neomycin and neomycin-derived antibiotics such as geneticin.The 3′ end of e) is linked to the 5′ end of a).

The pVex 1 vector was deposited on Apr. 16, 1999 at DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b,D-38124 Braunschweig, Germany, and was given accession number DSM 12776.

2. pDHFR Vector

The pDHFR vector confers resistance to ampicillin for selection inbacteria. This vector includes the cDNA coding for mice dihydrofolatereductase (DHFR), whose expression level is controlled by the SV40 virusearly promoter and its polyadenylation signal. The EPO expression levelachieved with the pVex 1-EPO (See Example 5) vector is enhanced severaltimes by the amplification of the DHFR and EPO genes in a culture mediumcontaining increasing concentrations of methotrexate (MTX).

The pDHFR vector was deposited on Apr. 16, 1999 at DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b,D-38124 Braunschweig, Germany, and was given accession number DSM 12777.

Example 5

Cloning of the EPO Coding Gene into an Expression Vector

The M13mp18 clone containing the EPO coding gene cloned as described inExample 3 was cut with Xho I-Hind III enzymes. The fragment thusobtained, of approximately 2.2 Kb, was isolated and cloned again usingthe same restriction sites of the pVex I vector. A positive pVex-EPOclone was isolated and analyzed showing that the EPO coding sequence SEQID NO:4 did not change during the cloning procedures.

The previous examples were performed according to conventional molecularbiology techniques. See Brown, “Gene Cloning”, (Chapman & Hall, London,England, 1995); Watson, et al., “Recombinant DNA, 2^(nd) Ed.”,(Scientific American Books, New York, N.Y., 1992); Sambrook et al,“Molecular Cloning. A Laboratory Manual”, (Cold Spring Harbor LaboratoryPress, 1989); Bishop et al., “Nucleic Acid and Protein Sequence. APractical Approach”, (IRL Press, 1987); Davis et al., “Basic Methods inMolecular Biology”, (Elsevier Science Publishing Co., New York, N.Y.,1986).

Example 6

Co-transfection and Amplification

A mutagenized CHO (Chinesc Hamster Ovary) cell line, deficient in theDHFR-enzyme gene (CHO-DHFR⁻), was used to facilitate gene amplificationwith MTX. During the entire process cells were grown in a 5% CO₂atmosphere at 37° C.

The CHO cells were cotransfected following the calcium phosphatetechnique which, using a 90 mm diameter Petri dish, was as follows:

(1) The culture medium (alpha-MEM, with 10% of fetal calf serum) wasreplaced with fresh medium 4-8 hours before transfection.

(2) 500 μL of 10 g/l HEPES (pH 7.1) solution, 500 μL of 16 g/l NaCl, 10μl of a 35 mM Na₂HPO₄ and 10 μl of 35 mM of NaH₂PO₄ solution were addedto a 5 ml test tube.

(3) In a separate 1.5 ml test tube, a solution with 60 μl of 2 M CaCl₂and 10 μg of each DNA vector to be transfected (pVex-EPO and pDHFR) wereadded. Water was added until a final volume of 500 μl was reached. ThepDHFR plasmid described in Example 2 is based on the pBR 322 plasmid,which is ampicillin-resistant. The pDHFR plasmid can be replicated in E.Coli and has the DHFR gene cloned between the SV40 early promoter andits terminator. The pDHFR plasmid codes for the expression of the DHFRprotein in CHO cells. This protein confers resistance to methotrexate,which can then be used to select cells showing high erythropoietinproductivity.

(4) The solution containing DNA and CaCl₂ was added drop by drop to thetest tube containing HEPES, while air was bubbled to obtain a rapidmixing and minimize local concentration. This method facilitated theformation of very small particles containing DNA and calcium phosphatewhich are more effectively incorporated by the cells.

(5) The solution was allowed to settle for 30 minutes and was added thento the Petri dish containing the cells.

(6) The solution was distributed among the cells by gentle shaking. Thecells were left overnight in an incubator under a 5% CO₂ atmosphere at37° C.

(7) Cells were rinsed twice with a PBS buffer (8 g NaCl; 0.2 g KCl; 1.44g Na₂HPO₄, 0.24 g NaH₂PO₄, water was added to 1 liter and pH wasadjusted to 7.4 with HCl). Fresh culture medium was then added.

The selection with geneticin (G 418) at a concentration of 600 g/mlbegun 24 hours after transfection. The cells which incorporated thepVex-EPO plasmid were able to resist the antibiotic, while all othersdied after 25 days. Resistant colonies were selected and theirproductivity was assayed. The three most productive clones were selectedfrom the isolated clones.

Taking advantage of the genetic constructions used in the invention, aselection was performed for each of the three clones using MTX assecondary selective agent at a 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M and 10⁻⁵ Mconcentration. For that purpose, the culture medium was changed toalpha-MEM without nucleotides, supplemented with 10% dialyzed fetal calfserum. It is essential to perform the dialysis process according to thefollowing protocol: 100 ml of serum were placed in a dialysis bag with a3,000 Da cut off (with a higher cut off, growth factors could be lost,and the cells would not be able to grow and reproduce), the bag washermetically closed, completely immersed in a container with 5 liters ofbidistilled water and left without agitation for 12 hours at 4° C. Afterthis step, the water was discarded and changed, leaving the bag to standfor an additional 12 hour period at 4° C. The dialysis bag was thenremoved and the serum recovered. The dialysis during shorter periods,with smaller volumes or without water replacement, would be ineffectivesince any trace of nucleotides in the serum will affect the MTXselection adversely. In the other hand, dialysis for longer periodswould also be ineffective because some proteins necessary for cellgrowth may precipitate preventing cell maturation.

Example 7

Isolation of High Productivity Clones

Clones that grew in 10⁻⁷ and 10⁻⁶ M of MTX were isolated and amplifiedin fresh alpha-MEM without nucleotides supplemented with 10% of dialyzedfetal calf serun. Once grown, the culture supernatant was assayed forthe production and secretion of EPO. For that purpose, a specificimmunoassay was used.

The process described above concluded with the selection of a clone ofrecombinant host cells producing 50,000 μg of erythropoietin/liter ofculture medium per day.

The recombinant host cell described in this example was deposited onApr. 16, 1999 at DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany andgiven accession number DSM ACC2397.

The effectiveness of transcription process in the cell was verified asdescribed in Example 8. The sequence of the obtained protein wasidentified following the procedure described in Example 9.

Example 8

Verification of the Specific Messenger RNA Sequence Produced by theReconmbinant Cells

1. Preparation of RNA from Cells

Total RNA was prepared from EPO producing cells, according to thefollowing protocol:

A 90 mm diameter Petri dish having confluent cells was washed twice with10 ml of PBS buffer. 2 ml of GTC buffer were then added and distributedevenly over the dish. The GTC buffer was composed of: 50 g guanidinumthiocyanate; 0.5 g N-Lauroylsarcosine, 2.5 ml 1 M sodium citrate (pH 7),0.7 ml β-mercapthoethanol, 0.33 ml of 30% antifoam agent (SIGMA) and 100ml H₂O q.s. (pH 7.0).

Cells were then Iysed. The lysate resulted in a highly viscous solution.The solution was transferred to a 15 ml test tube, and the process abovedescribed was repeated once more using 2 ml of GTC buffer.

The 15 ml test tube was vigorously stirred for 1 minute to break theDNA. A cesium chloride gradient was then performed. For that purpose, 4ml of a solution containing CsCl (95.97 g CsCl and 2.5 ml of 1 M Sodiumacetate, (pH 5.4), and water was added until a volume of 100 ml wasreached) were added to an ultracentrifuge test tube. Overthis solutionand without mixing, the suspension of the cells in GTC was then added.The test tube was next filled with GTC buffer and ultracentrifuged at31,000 rpm for 20 hours at 20° C.

During centrifugation, the RNA was deposited at the bottom of the testtube forming a pellet and the DNA obtained showed a band in the middleof the cesium chloride gradient. The supernatant was discarded toeliminate thoroughly the DNA. The RNA-containing pellet was let dry for5 minutes and was dissolved then in 200 μl of water and transferred to a1.5 ml test tube. 200 μl of 0.4 M Sodium acetate, (pH 4.8) and 2 volumesof ethanol were then added, the resulting solution was thoroughly mixedand left to settle for 30 minutes at −80° C. The solution was thencentrifuged in a microcentrifuge at 14,000 rpm for 15 minutes, thesupernatant was discarded and the precipitate was rinsed with 1 ml 80%ethanol. The pellet was dried and redissolved in 100 μl of water. Theconcentration of a 1:100 dilution of the RNA solution was measured at260 nm (1 OD unit corresponds approximately to 40 μg of RNA). All thesolutions and elements used were RNase-free.

2. Preparation of cDNA

Specific cDNA was prepared following the directions of a kit intendedfor that purpose (cDNA Synthesis System Plus, Amersham—cat. RPN 1256).The EPO 2 oligonucleotide was the primer used.

3. Cloning of cDNA Coding for EPO

Five percent of the cDNA thus obtained was amplified using 400 ng ofeach the EPO 2 and EPO 3 oligonucleotides, 2.5 mM of eachdeoxynucleotide in the proper buffer, and 2.5 units of Taq DNApolymerase, in a total volume of 100 μl. 35 amplification cycles wereperformed as follows: 1 minute at 93° C., 1 minute at 55° C. and 1minute at 72° C.

EPO 3 was synthesized as described for EPO 1 and EPO 2, and its sequence(5′ GAATTCCATGGGGGTGCACGAATGTCC 3′) (SEQ ID NO:5) corresponded to thefirst 20 bases coding for the EPO cDNA, and one site for the recognitionof the Eco RI enzyme. The Eco RI enzyme recognition site was added tofacilitate subsequent cloning steps.

A fragment of approximately 600 base pairs was obtained and cloned inM13mp18 and M13mp19 vectors. Using the Sanger's sequencing method, theinsert was sequenced in both directions to obtain the complete sequence.

Due to the high autocomplementarity of some regions of the gene, whichgives rise to many and very ambiguous compressions in theautoradiography, a sequencing kit using Taq DNA polymerase and modifiedbases was used. Lower quality results were obtained, but thecompressions were resolved. The kit used was the Pharmacia-LKBBiotechnology Gene aTaq.

The complete sequence of the human erythropoietin cDNA was isolated andcloned, showing to code for EPO. Consequently, the gene cloned in thecells and its transcription product were found complete and its sequencecorrect for EPO.

Example 9

Analysis of the EPO Produced

The EPO obtained by culturing the host cells as illustrated in thepreceding example was further purified to undergo various quality andidentificatory assays.

In a denaturing SDS-PAGE gel the EPO was identified as a wide band ofmolecular weight over 30 kDa as expected for EPO. See FIG. 1. The bandwas recognized by monoclonal and polyclonal antibodies against human EPOin a “Western blot” assay as expected for EPO. See FIG. 2. The treatmentwith glycanases proved the existence of the glycosidic chains in theextent and size as expected for EPO. See FIG. 3. The EPO produced wasshown to be composed of a series of species ranging isoelectric pointsfrom 3.0 to 4.5 as expected for EPO. See FIG. 4.

The complete amino acid sequence of the isolated protein, purified fromthe culture supernatant of transfected cell lines showed total homologywith natural human crythropoietin whose 165 amino acid sequence is asfollows (SEQ ID NO:1):

NH₂---Ala Pro Pro Arg Leu IIe Cys Asp       Ser Arg Val Leu Glu Arg TyrLeu       Leu Glu Ala Lys Glu Ala Glu Asn       IIe Thr Thr Gly Cys AlaGlu His       Cys Ser Leu Asn Glu Asn  Ile Thr       Val Pro Asp Thr LysVal Asn Phe       Tyr Ala Trp Lys Arg Met Glu Val       Gly Gln Gln AlaVal Glu Val Trp       Gln Gly Leu Ala Leu Leu Ser Glu       Ala Val LeuArg Gly Gln Ala Leu       Leu Val Asn  Ser Ser Gln Pro Trp       Glu ProLeu Gln Leu His Val Asp       Lys Ala Val Ser Gly Leu Arg Ser       LeuThr Thr Leu Leu Arg Ala Leu       Gly Ala Gln Lys Glu Ala IIe Ser      Pro Pro Asp Ala Ala Ser  Ala Ala       Pro Leu Arg Thr lIe Thr AlaAsp       Thr Phe Arg Lys Leu Phe Arg Val       Tyr Ser Asn Phe Leu ArgGly Lys       Leu Lys Leu Tyr Thr Gly Glu Ala       Cys Arg Thr GlyAsp----COOH

The presence of the four glycosilation sites along the 165 amino acidchain, as well as the complex carbohydrate structure, and in particular,the sialic acid terminal residues, which characterize EPO were verified.These results were further supported by a biological activity assay ofthe produced protein by an ex-hypoxic polycythemic mice test whichshowed complete concordance with the international EPO standard.

The productivity achieved, measured by a specific immunoassay, was 50 mgEPO per liter of culture medium per day.

All publications mentioned hereinabove are hereby incorporated in theirentirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

5 1 165 PRT Homo sapiens 1 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg ValLeu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile ThrThr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val ProAsp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly GlnGln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala ValLeu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp GluPro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser LeuThr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile SerPro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr AlaAsp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu ArgGly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr GlyAsp 165 2 29 DNA Artificial Sequence Description of Artificial Sequenceprimer 2 gaattctcga gatgggggtg cacggtgag 29 3 27 DNA Artificial SequenceDescription of Artificial Sequence primer 3 aagctttcat ctgtcccctgtcctgca 27 4 2164 DNA Homo sapiens 4 gaattctcga gatgggggtg cacggtgagtactcgcgggc tgggcgctcc cgccgcccgg 60 gtccctgttt gagcggggat ttagcgccccggctattggc caggaggtgg ctgggttcaa 120 ggaccggcga cttgtcaagg accccggaagggggaggggg gtggggcagc ctccacgtgc 180 cagcggggac ttgggggagt ccttggggatggcaaaaacc tgacctgtga aggggacaca 240 gtttgggggt tgaggggaag aaggtttgggggttctgctg tgccagtgga gaggaagctg 300 ataagctgat aacctgggcg ctggagccaccacttatctg ccagagggga agcctctgtc 360 acaccaggat tgaagtttgg ccggagaagtggatgctggt agctgggggt ggggtgtgca 420 cacggcagca ggattgaatg aaggccagggaggcagcacc tgagtgcttg catggttggg 480 gacaggaagg acgagctggg gcagagacgtggggatgaag gaagctgtcc ttccacagcc 540 acccttctcc ctccccgcct gactctcagcctggctatct gttctagaat gtcctgcctg 600 gctgtggctt ctcctgtccc tgctgtcgctccctctgggc ctcccagtcc tgggcgcccc 660 accacgcctc atctgtgaca gccgagtcctggagaggtac ctcttggagg ccaaggaggc 720 cgagaatatc acggtgagac cccttccccagcacattcca cagaactcac gctcagggct 780 tcagggaact cctcccagat ccaggaacctggcacttggt ttggggtgga gttgggaagc 840 tagacactgc ccccctacat aagaataagtctggtggccc caaaccatac ctggaaacta 900 ggcaaggagc aaagccagca gatcctacggcctgtgggcc agggccagag ccttcaggga 960 cccttgactc cccgggctgt gtgcatttcagacgggctgt gctgaacact gcagcttgaa 1020 tgagaatatc actgtcccag acaccaaagttaatttctat gcctggaaga ggatggaggt 1080 gagttccttt tttttttttt ttcctttcttttggagaatc tcatttgcga gcctgatttt 1140 ggatgaaagg gagaatgatc gggggaaaggtaaaatggag cagcagagat gaggctgcct 1200 gggcgcagag gctcacgtct ataatcccaggctgagatgg ccgagatggg agaattgctt 1260 gagccctgga gtttcagacc aacctaggcagcatagtgag atcccccatc tctacaaaca 1320 tttaaaaaaa ttagtcaggt gaagtggtgcatggtggtag tcccagatat ttggaaggct 1380 gaggcgggag gatcgcttga gcccaggaatttgaggctgc agtgagctgt gatcacacca 1440 ctgcactcca gcctcagtga cagagtgaggccctgtctca aaaaagaaaa gaaaaaagaa 1500 aaataatgag ggctgtatgg aatacattcattattcattc actcactcac tcactcattc 1560 attcattcat tcattcaaca agtcttattgcataccttct gtttgctcag cttggtgctt 1620 ggggctgctg aggggcagga gggagagggtgacatgggtc agctgactcc cagagtccac 1680 tccctgtagg tcgggcagca ggccgtagaagtctggcagg gcctggccct gctgtcggaa 1740 gctgtcctgc ggggccaggc cctgttggtcaactcttccc agccgtggga gcccctgcag 1800 ctgcatgtgg ataaagccgt cagtggccttcgcagcctca ccactctctt cgggctctgg 1860 gagcccaggt gagtaggagc ggacacttctgcttgccctt tctgtaagaa ggggagaagg 1920 gtcttgctaa ggagtacagg aactgtccgtattccttccc tttctgtggc actgcagcga 1980 cctcctgttt tctccttggc agaaggaagccatctcccct ccagatgcgg cctcagctgc 2040 tccactccga acaatcactg ctgacactttccgcaaactc ttccgagtct actccaattt 2100 cctccgggga aagctgaagc tgtacacaggggaggcctgc aggacagggg acagatgaaa 2160 gctt 2164 5 27 DNA ArtificialSequence Description of Artificial Sequence primer 5 gaattccatgggggtgcacg aatgtcc 27

What is claimed is:
 1. An isolated host cell comprising a vector whichcomprises: (a) a nucleotide sequence encoding the erythropoietinpolypeptide consisting of the amino acid sequence in SEQ ID NO:1 whereinsaid nucleotide sequence does not include non-coding fragments from 5′and 3′ regions; (b) a viral promoter; and (c) a viral terminator.
 2. Thehost cell deposited as DSM ACC2397.
 3. The host cell of claim 1, whereinsaid viral promoter and viral terminator comprises an early promoter andterminator of a SV40 virus.
 4. The host cell of claim 1, wherein saidvector comprises pVex 1 deposited as DSM
 12776. 5. The host cell ofclaim 1, further comprising a pDHFR vector.
 6. The host cell of claim 4,wherein said host cell is resistant to neomycin-derived antibiotics andmethotrexate.
 7. The host cell of claim 1, wherein said host cell is amammalian cell.
 8. The host cell of claim 7, wherein said mammalian cellis selected from the group consistng of CHO, COS, BHK, Namalwa, HeLa,Hep3B and Hep-G2 cells.
 9. The host cell of claim 1, wherein said hostcell comprises a CHO or COS cell.
 10. The host cell of claim 1, whereinsaid host cell comprises a CHO cell.
 11. A method for producing an EPOpolypeptide, comprising culturing the host cell of claim 1 underconditions such that said polypeptide is expressed and recovered. 12.The method of claim 11, wherein such conditions comprise exposure tomethotrexate.
 13. The method of claim 11, wherein said culture producesmore than 50 mg of EPO per liter of culture medium per day.